Laboratory-medical diagnostics constitutes an important basis for medical treatment. Due to the rising number of available diagnostic marker molecules, the possibilities of laboratory-medical diagnostics are being continuously expanded. Because of the volume of the detection reactions to be carried through, their great urgency, as well as for economical reasons, automated analysis methods, which are able to cope with a large number of different analyses within a short time, are employed with preference.
The raw material for the laboratory-medical analysis of a patient's state of health is typically a body fluid such as whole blood, plasma, serum, urine, ascites, amniotic fluid, saliva, liquor, etc., or tissue samples of different organs. The treating physician collects the sample from the patient, possibly subjects the same to a specific treatment, e.g. centrifugation, and then transfers the patient sample into a test tube, in which the sample is sent out or stored until the analysis is performed.
Depending on the parameter to be analysed, i.e. the components (analytes) to be analysed, the sample may either be stored at room temperature, or it has to be cooled, or stored in frozen condition. In the case of long-term storage, i.e. if the storage is for a period of several weeks, months or years, the patient samples must be frozen at −20° C. or at lower temperatures in order to prevent degradation of the analytes. The main cause for the degradation occurring at room temperature is the enzyme activities inherent in the liquid sample material.
Typical sample volumes for long-term storage amount to at least 500 μl. If, after a first analysis, further analytical determinations are to be made at a later point in time, or at various later points in time, several 500 μl samples have to be prepared starting from the original collection of blood, and stored, or stored in deep-frozen condition. These samples take up a relatively large space, which renders storage over a prolonged period of time relatively expensive. For this reason, long-term storage of patient samples is as a rule not applied. This, however, means that one has to forgo the possibility of at a later time falling back upon a sample collected earlier. In many cases, this is desirable or even necessary, namely when it is important to compare a patient's current condition, in terms of certain diagnostic parameters, with an earlier states of the same patient. As the case may be, it may be useful to record several earlier conditions in a patient at various points in time in order to perform a trend analysis for the relevant parameter. Such comparisons are, however, not possible where the relevant diagnostic test(s) were not, or could not be, made at the earlier point(s) in time in question and the original sample(s) was/were not stored.
For analysis of a patient's state of health, methods of the most different kind are currently utilized in laboratory medicine. Among these are first of all the activity determination of enzymes, special colouring reactions, immunochemical methods, cytological methods, and molecular-biological methods. In recent years, immunochemical methods have gained significance above all, and they have replaced many conventional methods. Molecular-biological methods are also increasingly making an entrance in routine diagnostics.
The currently utilized immunochemical analysis systems are based on antigen-antibody reactions, which mostly take place in a volume of ca. 10-500 μl. Here, the patient sample (body fluid, etc.), which contains the analyte to be detected, in this case an antigen, is incubated together with an antibody which is specific for this parameter and recognizes and binds to only this analyte. The product of this antigen-antibody reaction is a complex containing antibody-bound antigens. The higher the antigen concentration in a patient sample, the higher the concentration of antigen-antibody complexes formed. In current test systems, these antigen-antibody reactions take place either freely in solution (detection by turbidimetry or nephelometry), or they are performed on antigen-specific surfaces (e.g. RIA, ELISA). When the antigen-antibody reaction has taken place, in the first case the antigen-antibody complexes are in solution, in the second case they are bound to a solid phase, mostly a plastics surface. The detection and quantification of antigen-antibody complexes is performed in the case of turbidimetry or nephelometry by measuring the turbidity, in the case of RIA (radioimmunoassay) by radio-isotope-marked antibodies in conjunction with radiometric detection, in the case of ELISA (enzyme-linked immunosorbent assay) and LIA with enzyme-marked antibodies in conjunction with the detection of enzyme-catalysed colour-reactions. By means of these test systems, analyte and antigen concentrations can be detected in the range of up to 1 pg/ml (protein).
The miniaturization and automation of the above-mentioned analysis systems, which are established in laboratory technology, are currently intensively researched. Thus, in recent years, various miniaturized, solid-phase-linked test systems have been developed, which because of their small size are called “biochips” in analogy to computer chips. The size of such biochips is typically between 0.25 and 9 cm2. Biochips described in the literature consist of a solid matrix of glass (S. P. A. Fodor et al.: Light-directed, spatially addressable parallel chemical synthesis; Science 251 (February 1991), p. 767-773); C. A. Rowe et al.: An array immunosensor for simultaneous detection of clinical analytes; Analytical Chemistry Vol. 71 (Jan. 15, 1999) p. 433-439; L. G. Mendoza et al.: High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA); BioTechniques 27 (October 1999), p. 778-788), nylon or nitrocellulose membrane, silicone (J. Cheng et al.: Chip PCR. Nucleic Acids Research Vol. 24 (1996) p. 380-385), or silicon. Via different linker molecules, it is possible to covalently couple biomolecules, e.g. DNA, peptides or proteins, to those matrices. On a surface of 3.6 cm2 can be applied up to 10000 different biomolecules, which requires micrometer-accurate addressing of the biomolecules to special areas of the matrix which are separated from each other. This can be achieved, for example, by photolithographically controlled synthesis of the biomolecules (S. P. A. Fodor et al.: Light-directed, spatially addressable parallel chemical synthesis; Science 251 (February 1991), p. 767-773), or by spotting-on of the biomolecules by means of precision-mechanical, microprocessor-controlled programmable pipetting robots (L. G. Mendoza et al.: High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA); BioTechniques 27 (October 1999), p. 778-788; M. Eggers et al.: A microchip for quantitative detection of molecules utilizing luminescent and radioisotope reporter groups; BioTechniques 17 (1994) p. 516-523).
By means of the solid phase-bound biomolecules, the analytes to be analysed which are contained in the patient samples, can subsequently be bound and detected with suitable detection systems.
As detections systems of high sensitivity, there are employed, for instance, charge-coupled-device (CCD) cameras (L. G. Mendoza et al., M. Eggers et al.; loc cit.), phototransistors (T. Vo-Dinh et al.: DNA biochip using a phototransistor integrated circuit; Analytical Chemistry 71 (1999) p. 358-363), and fluorescence detectors (S. P. A. Fodor et al.; loc cit.).
The above-described biochips are currently being used exclusively for research purposes, e.g. for DNA sequencing, gene expression analysis, gene mutation analysis and protein-binding studies, i.e. antibody-binding studies.
In gene expression analysis (M. Schena et al.: Quantitative monitoring of gene expression patterns with a complementary DNA microarray; Science 270 (20 Oct. 1995) p. 467-470), DNA sequences which are complementary to certain genes are spotted on with the aid of a pipetting robot onto the matrix of a chip blank, with every single spot representing a certain gene. Subsequently, mRNA is isolated from the tissue sample to be analysed, marked with a fluorescent dye and applied on the entire biochip. Then, the binding of marked mRNA molecules to certain sites of the biochip can be detected. When the analysis has been carried through, the biochip is discarded as it is contaminated with the mRNA sample.
In a similar way, biochips can be utilized for DNA sequence analysis and for gene mutation analysis.
For immunochemical analysis of the sample material, Mendoza et al. (loc cit.) have described a prototype of a miniaturized test system. The authors used a special glass slide with 96 depressions (“wells”), each depression being printed, using a pipetting robot and a capillary printing method, with a pattern of spots consisting of 144 different antigens. After incubation with antibody-containing solution, those spots where antigen-antibody complexes have been formed can be detected by means of a CCD camera.
Hence, the biochips known from the prior art are restricted to such embodiments where specific, selected, or especially synthesized molecules are applied and bound to a solid matrix in a defined, miniaturized arrangement. These molecules serve to detect the presence of certain analytes in a heterogeneous mixture, e.g. the patient sample. This means that these biochips are test-specific, i.e. they allow only for those detection reactions which are pre-determined by the detection molecules present on the chip.
This approach has several disadvantages in respect of laboratory diagnostic applications. It is true that with this approach it is possible to perform a large number of different determinations at the same time; for a concrete problem, however, mostly only a limited number of parameters, i.e. analytes, is relevant. This means that the plurality of the detection molecules present on such a chip remains unused or is of no interest. In addition, one has to take into account that after incubation with the patient sample such a biochip can as a rule not be used for further analyses. For these reasons, the use of biochips of the type described is little suited for routine detection methods in laboratory medicine, and they can not be utilized in a cost-effective manner.
Furthermore, in the hitherto known biochips, new patient sample material is need for each further analysis to be carried out. The consequence is that the patient possibly has to appear several times for taking blood samples, which involves a certain effort both on the part of the physician and on that of the patient. If a certain examination is to be repeated at a later point using the sample material already collected, an intermediate storage of the sample material in liquid or; frozen condition will be necessary, which involves the above described drawbacks.
As a consequence of the progress in development, novel detection reactions are constantly being made accessible. To render the biochips known from the prior art usable for such newly developed detection reactions, the biochips would constantly have to be brought up to date, which involves high expenditure and, in addition, is only possible with a certain delay in time.
Often, at the time a new detection method becomes available, the original sample material of a certain patient is no longer available, so that it is mostly not possible to examine an alteration in time of the laboratory-medical parameter that is of interest, since the longer-term storage of patient sample material is problematic. The biochips known form the prior art cannot contribute to a solution of this problem.